Liedtke Carole M, Wang Xiangyun
Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106-4948, USA.
Biochemistry. 2006 Aug 29;45(34):10270-7. doi: 10.1021/bi0607249.
Regulation of the CFTR Cl channel function involves a protein complex of activated protein kinase Cepsilon (PKCepsilon) bound to RACK1, a receptor for activated C kinase, and RACK1 bound to the human Na(+)/H(+) exchanger regulatory factor (NHERF1) in human airway epithelial cells. Binding of NHERF1 to RACK1 is mediated via a NHERF1-PDZ1 domain. The goal of this study was to identify the binding motif for human NHERF1 on RACK1. We examined the site of binding of NHERF1 on RACK1 using peptides encoding the seven WD40 repeat units of human RACK1. One WD repeat peptide, WD5, directly binds NHERF1 and the PDZ1 domain with similar EC(50) values, blocks binding of recombinant RACK1 and NHERF1, and pulls down endogenous RACK1 from Calu-3 cell lysate in a dose-dependent manner. The remaining WD repeat peptides did not block RACK1-NHERF1 binding. An 11-amino acid peptide encoding a site on the PDZ1 domain blocks binding of the WD5 repeat peptide with the PDZ1 domain. An N-terminal 12-amino acid segment of the WD5 repeat peptide, which comprises the first of four antiparallel beta-strands, dose-dependently binds to the PDZ1 domain of NHERF1 and blocks binding of the PDZ1 domain to RACK1. These results suggest that the binding site might form a beta-turn with topology sufficient for binding of NHERF1. Our results also demonstrate binding of NHERF to RACK1 at the WD5 repeat, which is distinct from the PKCepsilon binding site on the WD6 repeat of RACK1.
囊性纤维化跨膜传导调节因子（CFTR）氯离子通道功能的调节涉及激活的蛋白激酶Cε（PKCε）与活化C激酶受体1（RACK1）结合形成的蛋白复合物，以及在人气道上皮细胞中RACK1与人钠/氢交换调节因子1（NHERF1）的结合。NHERF1与RACK1的结合是通过NHERF1的PDZ1结构域介导的。本研究的目的是确定人NHERF1在RACK1上的结合基序。我们使用编码人RACK1七个WD40重复单元的肽来研究NHERF1在RACK1上的结合位点。一种WD重复肽WD5，以相似的半数有效浓度（EC50）值直接结合NHERF1和PDZ1结构域，阻断重组RACK1和NHERF1的结合，并以剂量依赖的方式从Calu-3细胞裂解物中拉下内源性RACK1。其余的WD重复肽不阻断RACK1-NHERF1的结合。一个编码PDZ1结构域上一个位点的11个氨基酸的肽阻断WD5重复肽与PDZ1结构域的结合。WD5重复肽的N端12个氨基酸片段，包含四条反平行β链中的第一条，以剂量依赖的方式结合到NHERF1的PDZ1结构域，并阻断PDZ1结构域与RACK1的结合。这些结果表明，结合位点可能形成一个β转角，其拓扑结构足以结合NHERF1。我们的结果还证明了NHERF在WD5重复序列处与RACK1结合，这与RACK1的WD6重复序列上的PKCε结合位点不同。
